Project summary Neuronal activity can be modulated by transmitters that act through receptors coupled to heterotrimeric G proteins. Activation of these G protein signal transduction pathways may lead to the modulation of ion channels that alter neuronal excitability at the cellular level, ultimately causing changes in behavior at the organismal level. The long-term goal of our work is to identify and understand the mechanistic basis of neuromodulatory pathways. The goal of this proposal is to identify new regulatory pathways that lead to the modulation of a specific ion channel, the NALCN/NCA channel. The NALCN/NCA ion channel is a putative cation channel related to voltage-gated sodium and calcium channels, but whose precise cellular role and regulation are not well understood. However, mutations in NALCN or its associated subunits have been directly linked to human neurological diseases characterized by a range of symptoms, including abnormal movements and muscle contractions, intellectual disability, and seizures. Additionally, mutations in this channel in model organisms cause strong neuronal phenotypes including defects in rhythmic behaviors and neuronal excitability, demonstrating the physiological importance of this channel. Through a forward genetic screen in the nematode C. elegans, we found that the NCA ion channel is activated by a new signal transduction pathway acting downstream of the heterotrimeric G protein Gq. Activated Gq directly binds and stimulates the guanine nucleotide exchange activity of the Trio RhoGEF to activate the small G protein Rho, leading to the modulation of the NCA channel through unknown mechanisms. Here we will determine how Gq-Rho signaling modulates NCA activity by studying additional factors identified in our screen. In Aim 1, we will focus on the G protein- coupled receptor kinase GRK-2. Our genetic data support the hypothesis that GRK-2 modulates dopamine signaling that negatively regulates NCA activity through the Gq-Rho pathway. We will perform genetic, biochemical, cellular imaging, and electrophysiological experiments to determine how GRK-2 interacts with and affects the activity of dopamine receptors to eventually modulate the activity of the NCA channels. In Aim 2, we will focus on a mitogen-activated protein kinase (MAPK) pathway that modulates Gq-Rho activation of NCA. We will identify the members of this signaling pathway and determine how they modulate output of the Gq- Rho-NCA pathway. The proposed work is significant because it will identify the signaling pathways that modulate neuronal activity via a physiologically and medically important ion channel. The proposed work is innovative because it will close gaps in our understanding of how the NALCN/NCA ion channel is activated and identify new mechanisms of regulation of this channel.